CN101883860A - Biopolymer produced by fermenting the extract of soybean with folic acid and a composition containing thereof - Google Patents
Biopolymer produced by fermenting the extract of soybean with folic acid and a composition containing thereof Download PDFInfo
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- CN101883860A CN101883860A CN2008801188773A CN200880118877A CN101883860A CN 101883860 A CN101883860 A CN 101883860A CN 2008801188773 A CN2008801188773 A CN 2008801188773A CN 200880118877 A CN200880118877 A CN 200880118877A CN 101883860 A CN101883860 A CN 101883860A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
Abstract
Provided are a folyl extract of fermented soybean (EFS) produced by fermenting a culture including a folic acid and soybean extract by using a microorganism, and a composition including the folyl EFS. The folyl EFS has an anti-histamine effect, an anti-allergic effect, a calcium-absorption-promotion effect, a bone-growth-promotion effect, a cell growth promotion effect, a collagen biosynthesis promotion effect, a wrinkle improvement effect, and an UV-induced cell damage inhibition effect. Accordingly, the folyl EFS can be used in a skin external application or cosmetic composition, a health supplement food composition, a feed composition, and a pharmaceutical composition.
Description
Technical field
The present invention relates to a kind of leaf acyl (folyl) fermented soybean extract (extract offermented soybean that produces by the culture fermentation of using microorganism to make to comprise folic acid and soybean extraction, and the composition that comprises described leaf acyl EFS EFS).
Leaf acyl EFS of the present invention or composition have effect, the biosynthetic effect of acceleration collagen, the effect that improves wrinkle and the inhibition of the effect of antihistamine effect, anti-allergic effects, acceleration calcium absorption, the effect of quickening bone growth, the accelerated cell growth effect by ultraviolet ray (UV) radiation-induced cell injury.Therefore, leaf acyl EFS of the present invention or composition can be used in external preparation for skin product or make-up composition, health care supplementing food (health supplement food) composition, feed composition or the pharmaceutical compositions.
Background technology
Qing Betel sauce (Chungkookjang) and natto (Natto) are representative leavened foods, and are by using subtilis (Bacillus subtilis) that fermented soybean is made.When fermented soybean, produce gamma-polyglutamic acid-(γ-polyglutamate) and polyfructosan (polyfructan) polymkeric substance.Known gamma-polyglutamic acid-has preserves moisture and the cell activation function, and when with as safe plain cancer therapy drugs such as (Taxol) when using, make the effect increase of used medicine.Polyfructosan is a kind of food fibre, is used to fat-reducing, when throwing is given, makes stomach that satiety and depress appetite be arranged, and can prevent obesity thus.In addition, according to reports, polyfructosan suppresses harmful cell growth in the intestines, and helps bifidus bacillus (Bifidobacterium) growth (referring to Journal of Applied Microbiology, 92,5,958-963 page or leaf).
Comprise the patent (referring to No. the 0451631st, Korean registered patent) of relevant cosmetics material and the patent of relevant external preparation for skin product (referring to Korea S's publication 2001-0083876 number) about the patent of fermented soybean.It is that the enzyme process technology of 2,000 kilodaltons (kDa) or more gamma-polyglutamic acid-is (referring to App.﹠amp that people such as Seong mun-hoe have developed a kind of manufacturing molecular weight; Environ.Microbiol.2004; 70 (7), 4249-4255; With Korean registered patent 10-0399091 number).They have also registered a patent, and this patent is based on about xitix wherein in conjunction with having the result of study (Korean registered patent 10-0485727 number) of the material of this type of high-molecular weight gamma-polyglutamic acid-.Yet, still untappedly go out wherein that folic acid (claims vitamins B again
9) in conjunction with the fermented soybean polymkeric substance of a kind of novelty of gamma-polyglutamic acid-, with and use.
Known folic acid is a kind of material that reduces homocysteine content, and the content of homocysteine is relevant with dementia with the elderly's heart trouble.Therefore, when folic acid is used for the elderly's food, can obtain obvious effect.Folic acid is natural to be existed with the polyglutamic acid form, therefore is easy to be absorbed by animal and human's body.
Description of drawings
Fig. 1 is a series of micro-images, it shows the antihistamine effect of leaf acyl fermented soybean extract (leaf acyl EFS), wherein Figure 1A shows Normocellular micro-image, Figure 1B shows the micro-image of handling cell cell after 30 minutes with stimulator, and Fig. 1 C shows the micro-image of handling cell cell after 30 minutes with leaf acyl EFS and stimulator.
Fig. 2 shows that by quantitative histamine and Cromoglycic Acid (being known as the treating asthma agent) leaf acyl EFS suppresses the column diagram of the test result of histamine excretory effect.
Fig. 3 shows the column diagram of leaf acyl EFS about the test result of anaphylaxis (anaphylaxis), and the mortality ratio of each group is represented during with the peritoneal injection stimulator.
Fig. 4 is the column diagram that shows the test result of the calcium absorption promoter action of leaf acyl EFS in the animal small intestine, wherein in the experiment that obtains described result, animal is added with the feed of leaf acyl EFS, isolate the small intestine of animal and calcium amount of quantitatively respectively organizing, and to represent quantitative results with respect to the ratio of the group of not adding leaf acyl EFS in the given feed.
Fig. 5 is the column diagram of test result that shows the growth-promoting effect of leaf acyl EFS, wherein in the experiment that obtains described result, feed rat preset time section with leaf acyl EFS, get blood from each group, and utilize enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) insulin-like growth factor-i in the quantitative blood (insulin-like growth factor 1, IGF-1).
Fig. 6 is shown as fibrocellular primary culture and leaf acyl EFS or folic acid and cultivates the micro-image that obtains after 24 hours in inoblast substratum (fibroblast culture medium, FGM, KDR company).
Fig. 7 is shown as fibrocellular primary culture and leaf acyl EFS or folic acid to cultivate in fibroblastic growth substratum (FGM, KDR company) after 24 hours, by toluylene red (neutral red, NR) result's of the quantitative viable cell of test column diagram.
Fig. 8 shows that leaf acyl EFS promotes the result's of the biosynthetic effect of collagen column diagram, wherein in the experiment that obtains described result, use the biosynthesizing of fibroblastic primary culture promotion collagen and utilize the quantitative collagen of ELISA, and promote biosynthetic effect of collagen and transforming growth factor-beta (transforming growth factor beta, TGF-β) (be known as and promote the biosynthetic material of collagen) to promote the biosynthetic effect of collagen to compare leaf acyl EFS.
Fig. 9 shows the column diagram of testing the result of quantitative viable cell by NR, wherein in the experiment that obtains described result, the leaf acyl EFS that human keratinocyte's primary culture and folate content is about 10 weight % is at human keratinocyte's growth medium (keratinocyte growth medium, KGM, KDR company) cultivated about 72 hours in, with wavelength is the cell of ultraviolet ray (UV) the irradiation growth of 306 nanometers (nm), cultivates subsequently about 24 hours.
Embodiment
Technical problem
The invention provides a kind of fermented soybean of novelty, its characteristic good, low price, and can be used for various application.
Technical solution
For this reason, the present inventor attempts making the composition fermentation that comprises folic acid and soybean extraction, and makes novel leaf acyl fermented soybean extract (leaf acyl EFS).
Beneficial effect
Leaf acyl fermented soybean extract of the present invention (leaf acyl EFS) inhibition mastocyte produces histamine, and wherein mastocyte produces a kind of symptom that the phenomenon of histamine is people's appearance of the serious skin adverse reaction of generation (for example irritated (allergy)).Leaf acyl EFS is also very effective to alleviating anaphylaxis.In addition, leaf acyl EFS promotes in the small intestine as the absorption of mineral substance such as calcium, and promotes Mammals femur (Thigh bone) to grow.In addition, leaf acyl EFS is because of having the cell activation function, and promotes the biosynthesizing of collagen, and when being applied to human skin, the effect of the wrinkle that can be improved can also obtain other various effects thus thus, for example preserves moisture and anti-allergic effects.And leaf acyl EFS also protects cell in order to avoid by ultraviolet ray (UV) radiation injury.Therefore, leaf acyl EFS and the composition that comprises leaf acyl EFS can be used for keeping healthy in supplementing food, makeup, external preparation for skin product, pharmaceutical product and the animal feed.In addition, compare with the other products with similar effect, the production cost of leaf acyl EFS of the present invention is lower, and productive rate is higher.
The invention pattern
The present invention is directed to a kind of leaf acyl fermented soybean extract (leaf acyl EFS) and its application that produces by the culture fermentation of using microorganism to make to comprise folic acid and soybean extraction.
When using microorganism to make fermented soybean, produce gamma-polyglutamic acid-, it is a kind of amido peptide connection (peptide-linked) of L-glutamic acid polymkeric substance to γ-carboxyl.Simultaneously, folic acid (vitamins B 9 or VitB11 (pteolyl glutamic acid)) is L-glutamic acid and pteroyl ring (pteolyl ring) bonded L-glutamic acid analogue.The present inventor identifies; the conventional gamma-polyglutamic acid-that produces when fermented soybean can be produced by the synthetic enzyme that genus bacillus (Bacillus) microorganism belonging to genus produces; and, test according to synthetic enzyme tends to make the folic acid polymeric fact when contacting with folic acid (wherein pteroyl loops connected valleys propylhomoserin) when making synthetic enzyme.Thus, the present inventor finds, adds folic acid when using microorganism to make fermented soybean, folic acid will be incorporated in the gamma-polyglutamic acid-by the enzyme of microorganisms, and can produce a kind of new polymkeric substance in a large number thus, that is, comprise the leaf acyl polyglutamic acid of folic acid and L-glutamic acid.Correspondingly, the invention provides a kind of leaf acyl EFS and a large amount of method that produces leaf acyl EFS.Leaf acyl EFS of the present invention comprises leaf acyl polyglutamic acid.The Polylevulosan (levan) (microorganism polyfructosan) that leaf acyl EFS of the present invention is produced in the time of also can comprising fermented soybean.
The folic acid of incorporating among the leaf acyl EFS is very different with the characteristic of folic acid itself, and has high water-solublely, and can more effectively be used for industrial application thus, and be easier to the little intestinal absorption into animal.
In addition, because the Polylevulosan (microorganism polyfructosan) that leaf acyl EFS of the present invention produced in the time of can also comprising fermented soybean, so can promote the growth of bifidus (bifidus lactobacillus), and can obtain the stomach and intestine cleaning action thus and prevent fat effect.
Leaf acyl EFS of the present invention also has and proportional higher immunostimulation of the concentration of leaf acyl EFS and anti-allergic effects, and this differentiates by external (in vitro) test and animal experiment.Folic acid itself does not have described immunostimulation and anti-allergic effects.Leaf acyl EFS of the present invention suppresses histamine secretion (referring to Fig. 1 and Fig. 2) and alleviates anaphylaxis (referring to Fig. 3).Therefore, leaf acyl EFS of the present invention can be used in the food, preventing baby and children acute allergy, and improves adult's physique (constitution).In addition, leaf acyl EFS of the present invention can be used in external preparation for skin product or the make-up composition, to alleviate atopic dermatitis (atopic dermatitis) or allergy.
Leaf acyl EFS of the present invention promotes the biosynthesizing (referring to Fig. 6) of collagen, and has the effect (referring to table 5) that improves wrinkle.Therefore, leaf acyl EFS of the present invention can be used in the exterior-applied article or make-up composition of preserving moisture, improve skin based on metabolism activation, and has the characteristic of improving wrinkle and the effect that causes thus.
Leaf acyl EFS of the present invention promotes the calcium absorption (referring to Fig. 4) of animal small intestine.Therefore, leaf acyl EFS can be with using as medicinal metals such as calcium, iron or manganese, and can produce various products thus, and it also comprises medicine except that comprising functional product, and for example nutritious supplementary, pregnant woman are with anti-anemic or diabetes supplement.
In addition, leaf acyl EFS of the present invention promotes bone growth (referring to table 4), and increases the somatomedin (IGF-10) (referring to Fig. 5) in the blood.The example of current available growth helping matter is KI-180, and it is by the common research and development in Highki growth clinic (Highki growth clinic) and korean foods institute (Korea FoodResearch Institute).Because KI-180 extracts from herbal medicine, thus its cost an arm and a leg, and therefore be used to costliness medicine and in the non-food product.Thereby, be difficult to the KI-180 commercialization.Yet, because leaf acyl EFS of the present invention can produce in a large number by microbial fermentation, so leaf acyl EFS can be used for multiple application, for example be suitable for children functional foodstuff, be suitable for the functional foodstuff of postmenopausal women's preventing osteoporosis disease and nutritious supplementary.Thus, leaf acyl EFS can promote the easy commercialization of material of growth than routine.
According to above-mentioned experimental result, the invention provides:
[1] a kind of leaf acyl EFS comprises folic acid polyglutamic acid (folic acid polyglutamate) as main ingredient, and wherein said leaf acyl EFS produces the culture fermentation that comprises folic acid and soybean extraction by the use microorganism;
[2] according to [1] described leaf acyl EFS, wherein said microorganism is selected from genus bacillus (bacillus) microorganism belonging to genus or excellent bacillus (corynebacterium) microorganism belonging to genus;
[3] according to [1] described leaf acyl EFS, wherein said microorganism is subtilis (Bacillussubtilis);
[4] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has antihistamine effect;
[5] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has anti-allergic effects;
[6] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has the effect that promotes calcium absorption;
[7] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has the effect that promotes bone growth;
[8] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has the biosynthetic effect of the collagen of promotion;
[9] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has the effect that improves wrinkle;
[10] according to each described leaf acyl EFS in [1] to [3], wherein said leaf acyl EFS has the effect that promotes the cell growth;
[11] a kind of composition that is used for external preparation for skin product or makeup, it comprises according to each described leaf acyl EFS in [1] to [3] as active ingredient;
[12] according to [11] described composition that is used for external preparation for skin product or makeup, wherein said composition is used for antianaphylaxis or improves atopic dermatitis;
[13] according to [11] described composition that is used for external preparation for skin product or makeup, wherein said composition be used to activate skin cells metabolism, prevent wrinkle formation or improve wrinkled appearance;
[14] according to [11] described composition that is used for external preparation for skin product or makeup, wherein said composition is used for suppressing by ultraviolet ray (UV) radiation-induced cell injury;
[15] a kind of health care supplementing food composition, it comprises according to each described leaf acyl EFS in [1] to [3] as active ingredient;
[16] according to [15] described health care supplementing food composition, wherein said health care supplementing food composition is to be used for drink;
[17] according to [15] described health care supplementing food composition, wherein said health care supplementing food composition has the effect that promotes growth;
[18] according to [15] described health care supplementing food composition, the effect that wherein said health care supplementing food composition has the osteoporosis of preventing;
[19] according to [15] described health care supplementing food composition, wherein said health care supplementing food composition has the effect that promotes calcium absorption;
[20] according to [15] described health care supplementing food composition, wherein said health care supplementing food composition prevents allergy or improves physique;
[21] a kind of feed composition, it comprises according to each described leaf acyl EFS in [1] to [3] as active ingredient;
[22] a kind of medicinal compositions that promotes the cell growth, it comprises according to each described leaf acyl EFS in [1] to [3] as active ingredient;
[23] medicinal compositions of a kind of treatment or preventing osteoporosis disease, it comprises according to each described leaf acyl EFS in [1] to [3] as active ingredient;
[24] a kind of medicinal compositions that promotes calcium absorption, it comprises according to each described leaf acyl EFS in [1] to [3] as active ingredient; With
[25] method of a kind of manufacturing leaf acyl fermented soybean extract (EFS), it is by the use microorganism culture fermentation that comprises folic acid and soybean extraction to be carried out.
Leaf acyl EFS of the present invention can use the soybean extraction manufacturing.Now describe the method for making soybean extraction in detail.Soybean is added in the distilled water, and wherein the ratio of soybean and distilled water is about 0.1% to about 50%.100 ℃ of following heated mixt 1 to 2 hour, and slowly cooling, from wherein taking out solid contents, produce soybean extraction thus fully subsequently.Use 0.1% to 25% glucose, 0.01% to 10% Sodium phosphate dibasic (Na
2HPO
4) and 0.01% to 10% SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
4) the preparation substratum.Subsequently, 0.1% to 90% soybean extraction and 0.01% to 10% folic acid are added in the substratum, with the gained medium sterilization, then the subtilis that will separate from food is inoculated in the substratum, and under 10 ℃ to 60 ℃ temperature, particularly 35 ℃ under 55 ℃, in pH 5.5 to pH 8.5 times, in facultative fermentation condition, carry out zymotechnique, continue 48 hours to 96 hours.After zymotechnique is finished, remove insoluble material by continuously centrifuged, for example cell, and use 2M hydrochloric acid is controlled at pH 3 to pH 5 with the pH value of gained solution, and pH 4 particularly.Then, to wherein adding ethanol, wherein the alcoholic acid amount is 2 times or more times of pH value Be Controlled solution.Obtain leaf acyl EFS thus, freeze-drying is also preserved with powder type.Use ordinary method, carry out grouping according to molecular weight.For example, can utilize chromatography or use cross-flow ultra filtration unit (cross-flow ultrafiltration unit) to carry out grouping.
The microorganism that can be used for folic acid and the used soybean extraction of the present invention are fermented can be bacillus micro-organism or Corynebacterium microorganism, and it can produce gamma-polyglutamic acid-.The example of bacillus micro-organism comprises subtilis, Bacillus anthracis (Bacillus anthracis), Bacillus licheniformis (Bacillus licheniformis) and bacillus megaterium (Bacillus megaterium).For example, bacillus micro-organism can be a subtilis.The Corynebacterium microorganism can be Corynebacterium glutamicum (Corynebacterium glutamicum), but is not limited thereto.
In addition, leaf acyl EFS has anti-allergic effects, and promotes to comprise the people in the growth of interior animal and the growth of bifidus bacillus and Bacterium lacticum (Lactobacillus), produces high livestock income and high meat thus.Therefore, leaf acyl EFS can be used as fodder additives in animal feed industries.
Composition of the present invention can comprise leaf acyl EFS, and the amount of its middle period acyl EFS can account for composition 0.01 weight % to 60 weight %, and 0.1 weight % is to 50 weight % particularly.
In make-up composition of the present invention, leaf acyl EFS can used component use in an amount of conventional skin cosmetics material.The example of this type of component comprises oil plant, water, tensio-active agent, wetting agent, lower alcohol, thickening material, sequestrant, tinting material, sanitas and spices.The make-up composition that the present invention comprises leaf acyl EFS can be used in moisture-keeping cosmetics, facial cleansing product or other makeup, but is not limited thereto.Particularly, the make-up composition that the present invention comprises leaf acyl EFS can be used in the makeup, for example astringent lotion (astringent cosmetic water), gentle skin astringent (emollient cosmeticwater), nutrition astringent (nutrient cosmetic water), various breast frost, essential oil, facial mask (pack) or foundation cream (foundation); Cleaning product; The facial cleansing product; Soap; Maintenance product (treatment); Or skin lotion (cosmetic solution).More specifically get on very well, the make-up composition that the present invention comprises leaf acyl EFS can be used for moisturizer (skin lotion), gentle skin liquid (skin softener), transfer skin liquid (skintoner), convergence emulsion (astringent lotion), milk emulsion (milk lotion), moisturizing emulsion (moisturizing lotion), nutritional emulsions (nutrient lotion), massage cream frost (massage cream), nutrition breast frost (nutrient cream), moisturizer frost (moisturizing cream), profit hand breast frost (handcream), essential oil, the nutrition essential oil, facial mask, soap, shampoo, cleaning foam, cleaning breast (cleansinglotion), cleansing cream (cleansing cream), health breast (body lotion), clean skin liquid (bodycleanser), oil plant, muffin (press powder), in loose powder or the eye shadow.
Medicinal compositions of the present invention can solid, semisolid, solution or oil form use, or uses with dispersion agent, micella or liposome form.Medicinal compositions can comprise: the organic or inorganic supporting agent or the vehicle that are suitable for intestines or non-oral administration medicine supplying; And leaf acyl EFS of the present invention is as activeconstituents.Active ingredient can the conventional nontoxic pharmaceutically acceptable supporting agent of tablet, pill, capsule, suppository, pharmaceutical solutions, oil formulation, suspension preparation, salve or easy-to-use arbitrary class preparation mixes with for example being suitable for.The tri-glyceride that available supporting agent can be glucose, lactose, gum arabic, gelatin, N.F,USP MANNITOL, starch paste, Magnesium Trisilicate salt, talcum, W-Gum, Keratin sulfate, silica colloidal, yam starch, urea, have medium chain, or being suitable for making arbitrary class supporting agent of preparation, it can be solid, semisolid or liquid.Medicinal compositions can further comprise supplement, stablizer, thickening material, tinting material or spices.The medicinal compositions that comprises active ingredient can form and be easy to oral any preparation, but for example tablet, lozenge, rhombus tablet, water-based or oily suspensions preparation dispersion powder, granule, oil, hard or soft capsule, syrup or elixir.Can use to be used for making medicinal compositions and the known any method of this technology, prepare these composition for oral administration.For obtaining good medicinal characteristic and make the taste of these composition for oral administration more attractive, described composition for oral administration can further comprise at least a functional agent that is selected from by the following group that forms: sweeting agent, for example sucrose, lactose or asccharin; Seasonings, for example peppermint, Pyrola Herb (pyrola japonica) or cherry; Tinting material; And sanitas.Can use ordinary method preparation to comprise the tablet of active ingredient and nontoxic pharmaceutically acceptable vehicle.Nontoxic pharmaceutically acceptable vehicle can be: inactive excipients, for example lime carbonate, lactose, calcium phosphate or sodium phosphate; Granulating agent or decomposition agent, for example W-Gum, yam starch or Lalgine; Tackiness agent, for example tragacanth (tragerkans rub), corn potato, gelatin or gum arabic (Acasia); Or lubricant, for example hard magnesium, stearic acid or talcum.Tablet can not coat dressing, maybe can use ordinary method to coat dressing with decomposition or the absorption of delay tablet in stomach and intestine, and makes drug effect continue one long period thus.The delay material that is used for this purpose can be glyceryl monostearate or distearin.These materials can also be used to form osmotic therapeutic tablets, with emitting of control active ingredient.
In some cases, oral preparations can be a hard gelatin capsule, wherein active ingredient with mix as lime carbonate, calcium phosphate or kaolin non-activity solid diluents such as (kaolin); Or soft gelatin capsule, wherein active ingredient is mixed with water or as peanut oil, whiteruss or olive wet goods oil medium.
Because individuality to be treated has multiple symptom, and medicine has the particular treatment characteristic, so Ying You expert measures and individually changes dispensing dosage to the reaction of treatment and according to reacting.Usually, the dispensing dosage of every day can be that about 10 micrograms of per kilogram of body weight arrive about 200 milligrams, and particularly, about 50 micrograms of per kilogram of body weight are to about 100 milligrams.The dispensing frequency of every day can be for 4 times or still less.
The food compositions that comprises leaf acyl EFS of the present invention can be used for making various food.The example of this based food comprises meat, sausage, bread, chocolate, candy, dessert, confectionery (confectionary), za (pizza), various noodles, chewing gum, milk-product (comprising ice-creams), various soup, beverage, tea, drink, wine and vitamin complex, but the invention is not restricted to this.
With reference to following example and experiment embodiment the present invention is described in more detail.These examples and experiment embodiment are for purposes of illustration, and do not plan to limit the scope of the invention.
＜example 1 〉
Make leaf acyl EFS
In order to make leaf acyl fermented soybean extract (leaf acyl EFS), at first prepare soybean extraction.Specifically, soybean is added in the distilled water, wherein the ratio of soybean and distilled water is 50%, and in 100 ℃ of following heated mixt 1 hour and slowly cooling, from wherein taking out solid contents, produces soybean extraction thus subsequently.Use 2.0% glucose, 1.0%Na
2HPO
4And 0.5%NaH
2PO
4The preparation substratum.Subsequently, 90% soybean extraction and 0.5% folic acid are added in the substratum, with the gained medium sterilization, the subtilis that will separate from food is inoculated in the substratum subsequently, and carries out zymotechnique under 45 ℃ of temperature, continues 72 hours.After zymotechnique is finished, remove insoluble material by continuously centrifuged, for example cell, and use 2M hydrochloric acid is controlled at pH 4 with the pH value of gained solution.Then, to wherein adding ethanol, wherein the alcoholic acid amount is 2 times or more times of pH value Be Controlled solution.Obtain leaf acyl EFS thus, freeze-drying is also preserved with powder type.In order to prepare the polymkeric substance that molecular weight is 1000 kilodaltons, using sepharose (Sepharose) 4B to remove molecular weight is 500 kilodaltons or lower polymkeric substance, and use cross-flow ultra filtration unit (making) gained solution to be divided into to contain molecular weight be 1 by Vivascience company, the part of 000 kilodalton or higher polymkeric substance, and contain the part that molecular weight is lower than the polymkeric substance of 1,000 kilodalton.By gel permeation chromatography (gel permeationchromatography, GPC), use liquid chromatography (Liquid chromatography, LC) measure that to have molecular weight be 1, the molecular-weight average of the part of 000 kilodalton or higher polymkeric substance, and measured molecular-weight average is 2,500 kilodaltons.With this two portions drying, and use identical amount of powder.
＜example 2 〉
Manufacturing comprises the composition of leaf acyl EFS
Preparation comprises the leaf acyl EFS of 0.0 weight % preparation in the 10.0 weight % examples 1 and can be used for the various compositions of the additive in food and the medicine.
(1) preparation example 1: the food compositions that comprises leaf acyl EFS
Preparation comprises the food compositions of component shown in the table 1 and its amount, to make drinkable health care supplementing food.In these compositions,, suitably control the amount of Purified Water according to the amount of natural antiseptic agent.
[table 1] comprises the food compositions (weight %) of leaf acyl EFS
| | Composition | 1 | Composition 2 | Composition 3 | Composition 4 | Composition 5 | Composition 6 |
| Leaf acyl EFS Cromoglycic Acid sodium salt (cromoline sodium salt) cyclodextrin trace mineral supplement, for example the mineral substance distilled water natural antiseptic agent of preparation in the example 1 | --2 3 95 is an amount of | -123 94 is an amount of | 0.05-2 3 94 is an amount of | 0.1-2 3 94 is an amount of | 0.2-2 3 94 is an amount of | 0.3-2 3 94 is an amount of |
(2) preparation example 2: the make-up composition that comprises leaf acyl EFS
Preparation comprises the make-up composition of the component shown in the table 2 and its amount and comprises the reference composition that distilled water substitutes leaf acyl EFS.In table 2, (A) represent water-soluble substances, and (B) represent oil-soluble substance.According to the amount of sanitas, food flavouring and pigment, suitably control the amount of sterile distilled water.
[table 2] comprises the make-up composition (weight %) of leaf acyl EFS
| | Composition | 1 | Composition 2 | |
| ??A | The leaf |
??5.0??-??4.0??4.0??68.5 | ??5.0??1.0??4.0??4.0??67.5 | |
| ??B | Ethanol hexadecanol beeswax Vaseline (Vaseline) squalene dimethyl polysiloxane POE (10) monoleate glyceryl monostearate sanitas food flavouring pigment | 5.0 1.0 0.5 2.0 6.0 2.0 1.0 1.0 is an amount of an amount of | 5.0 1.0 0.5 2.0 6.0 2.0 1.0 1.0 is an amount of an amount of |
(3) preparation example 3: the feed composition that comprises leaf acyl EFS
Preparation comprises the feed composition of the component shown in the table 3 and its amount.
[table 3] comprises the feed composition (weight %) of leaf acyl EFS
| | Composition | 1 | Composition 2 |
| The leaf acyl EFS lime carbonate zeolite of preparation in the example 1 | ??-??70??30 | ??1??70??29 |
＜example 3〉the antianaphylaxis test
Example 3-1: differentiate antihistamine effect by the microscopic examination mastocyte
In order to differentiate the antihistamine effect of leaf acyl EFS, with test materials be applied to mastocyte (ATCCTIB-64, P815), and by the microscopic examination mastocyte (referring to people such as George Chiang, TheJournal of Urology, December, 164,2000 years, the 2119-2125 page or leaf).Figure 1A shows the image of normal mastocyte; Figure 1B shows the image of handling and leave standstill 30 minutes mastocyte with 10 mcg/ml C48/80 (sigma company) as stimulator; And Fig. 1 C shows the image of handling and leave standstill 30 minutes mastocyte with leaf acyl EFS and stimulator.To Fig. 1 C, normal mastocyte is round and smooth referring to Figure 1A.When immune response takes place when, the mastocyte roughen.Yet, when handling mastocyte and leaving standstill 15 minutes as stimulator, have projection on mastocyte distortion and its surface, and after past 30 minutes, mastocyte loses its original shape fully and breaks, and secretes histamine thus with C48/80.The mastocyte surface deformation is that C48/80 stimulates the cytolemma of mastocyte and secretes various signal delivery materials and cell membrane stability attenuating, makes the unsettled phenomenon of surface of cell membrane thus.Therefore, after can handling according to C48/80, the degree that the initial surface shape of mastocyte is kept is differentiated antihistamine effect.Yet when handling with leaf acyl EFS and leaving standstill 15 minutes, mastocyte keeps its original shape, and even 30 minutes in the past after, only the emiocytosis histamine of minority and its membranolysis.
Example 3-2: the histamine by quantitative mastocyte is differentiated antihistamine effect
Carry out this experiment and be in order to differentiate that leaf acyl EFS reduces the degree of histamine amount secreted when handling mastocyte with stimulator.Use the amount of the quantitatively secreted histamine of ELISA.With 1% Sodium Cromoglicate (being known as the treating asthma medicine) as control group, and C48/80 as stimulator induce the mastocyte activation (ATCC TIB-64, P815).Fig. 2 is the column diagram that shows experimental result.
Referring to Fig. 2, when using about 0.5% during as the Sodium Cromoglicate for the treatment of asthma medicine, histamine excretory inhibiting rate is 50%, and ED50 (50% effective dose) value is 0.5% thus; And when using leaf acyl EFS, the ED50 value is about 0.063%, 1/8 when being to use Sodium Cromoglicate.As indicated above, as can be seen, leaf acyl EFS has good antihistamine (antianaphylaxis) effect.
Example 3-3: antianaphylaxis (antiallergic property reaction) test
For the antihistamine effect (in example 3-1, differentiating) of confirming leaf acyl EFS, fed mouse 7 days with compositions for use 1-6 in the preparation example 1, subsequently it is reacted to induce antiallergic property through peritoneal injection C48/80.Fig. 3 is the column diagram that shows experimental result.
C48/80 be a kind of when through peritoneal injection immune stimulatory organ and in 30 minutes, induce the material of acute anaphylaxis.In this experiment, count out behind the peritoneal injection C48/80 quantity of test mice dead in the section (24 hours) at the fixed time.In this experiment, every group is used 10 mouse (BALB/C, male), and respectively group is tested 3 times, and the mean value of three experimental results is expressed as mortality ratio (%).
Referring to Fig. 3, using the mortality ratio of the group of composition 1 nursing of adding is 93.3%, in 10 mouse 9 death is arranged in 24 hours behind injection C48/80.The mortality ratio (that is the mortality ratio of control group) of the group of feeding with the composition 2 that adds Sodium Cromoglicate is 20%.Experimental group uses the composition 3 to 6 of the various concentration leaf acyl EFS of interpolation 0.05% to 0.3% to feed respectively.The experimental group mortality ratio of adding the composition 3 of 0.05% leaf acyl EFS is 33.3%, and the mortality ratio of composition 4 is 20%, and the mortality ratio of composition 5 is 6.7%, and the mortality ratio of composition 6 is 16.7%.In view of these results, 1% inhibition that anaphylaxis is taken place as the Sodium Cromoglicate for the treatment of asthma medicine is up to 80% as can be seen, and the concentration inhibition that to be 0.2% leaf acyl EFS take place anaphylaxis is up to 94%.Yet unusual anus swelling appears in the mouse that survives in 24 hours behind the injection stimulator C48/80.Comprise the mouse of the composition 2 of Sodium Cromoglicate at about 80% feeding, and when using the composition 3 to 6 that comprises various concentration leaf acyl EFS respectively, in about mouse of 10%, 0%, 0% and 0%, find this incident.Therefore, as can be seen, Sodium Cromoglicate suppresses the generation of anaphylaxis, and prevents dead mouse, but does not suppress the sequela of anaphylaxis, and leaf acyl EFS suppresses the sequela and the dead mouse of anaphylaxis.
＜example 4〉promote the effect of calcium absorption
Use mouse (BALB/C, male) to carry out this experiment, and every day twice, make oral each feed of mouse.The oral phase is 3 days, and at oral after date, isolates the small intestine of mouse.Wash the gained small intestine 3 times or more times with physiological saline, and organize isolating small intestine from each with teflon homogenizer (teflon homogenizer) grinding.Centrifugal products therefrom, and separation of supernatant.Subsequently, use calcium quantification kit (Wako company) to measure the calcium amount that absorbs in the small intestine.Fig. 4 is the column diagram that shows experimental result.
Referring to Fig. 4, to compare with the control group of the feed that gives to add, the calcium absorptivity of group that is added with the feed of leaf acyl EFS increases about 84% to 96%.The calcium absorptivity of group that is added with the feed of calcium salt is about 4.7%, and (casein phosphopeptide, the group of feed CPP) is about 21%, and the group that is added with the feed of gamma-polyglutamic acid-is about 56.8% to be added with phosphopeptide caseinate.For leaf acyl EFS, when molecular weight is 500 to 1,000 kilodaltons, height when calcium absorptivity is higher than 1,000 kilodalton than molecular weight.This possibility of result is owing to when the molecular weight of leaf acyl EFS is too high, calcium ion (a kind of divalent ion) and electronegative leaf acyl EFS reaction, and may produce insoluble precipitation thus.Therefore, when the molecular weight of leaf acyl EFS is higher, it is desirable to produce soluble calcium-Ye acyl EFS mixture.
＜example 5〉promote the effect of growth
Because as example 3 described in, leaf acyl EFS promotes calcium absorption in intestines, thus this experiment of execution absorbs with discriminating whether calcium in the intestines influences that animal skeleton is grown and blood in somatomedin (IGF-1).Mammals is added with 2 weeks of feed of leaf acyl EFS, and measures the length and the weight of Mammals femur (Thigh bone), and get blood and measure growth factors I GF-1.The result is compared with the control group of the feed that is added with physiological saline.The Mammals of test is the rat (SD rat, male) in 4 ages in week.Comparative group is given by P﹠amp; " the true long unit (Kikerup gold) that generates " product that B company makes, it contains slender acanthopanax (Acantopanax), Semen Flos Carthami (red flower seed), Radix Rehmanniae Preparata (Rehmanniaeradix preparata), Poria cocos (Poria cocos), Chinese yam (Dioscoreae rhizoma), skunk bush (Corni fructus), deer horn Chinese medical extracts such as (antler) as main component, and tests under condition identical when using leaf acyl EFS.
The result is as shown in table 4 and Fig. 5.
When standard error is 5% or when lower, the mean length of the femur (Thigh bone) of the control group of feeding with physiological saline is 29.22 millimeters, uses P﹠amp; The mean length of the femur (Thigh bone) of the comparative group that the promotes growth product of B company is fed is 29.63 millimeters, and is 30.04 millimeters with the mean length of the femur (Thigh bone) of the experimental group of leaf acyl EFS nursing.Compare with control group, comparative group has about 1.4% growth promoting function, and experimental group has about 3.1% growth promoting function (table 4).Utilize the quantitative IGF-1 of ELISA (being known as the positive growth factor in the blood).Thus, control group has 1409.5 mcg/ml IGF-1, and comparative group has 1566 mcg/ml IGF-1, and experimental group has 1637.1 mcg/ml IGF-1.That is to say that the IGF-1 in the comparative group is higher by 11.1% than the IGF-1 in the control group, and the IGF-1 in the experimental group is than the IGF-1 in the control group high by 16.2% (Fig. 5).The weight of comparative group and experimental group femur (Thigh bone) is compared respectively according to group high about 1.3% and about 2.3%.In view of these results, as can be seen, leaf acyl EFS has the effect that promotes growth of animal.In addition, when with P﹠amp; When the promotes growth production cost of products of B company is compared with leaf acyl EFS, consider P﹠amp; The promotes growth product of B (the true length unit that generates) contains VITAMIN and nutrient substance, and the production cost of leaf acyl EFS is about 1/20 of a described promotes growth product.Therefore, from comprising production cost, infer leaf acyl EFS and have excellent characteristic in interior all respects.
The promotion bone growth effect of table 4. folic acid
| Test | Length (millimeter) | Epimere width (millimeter) | Intermediate width (millimeter) | Hypomere width (millimeter) | Weight (milligram) |
| Control group | ??29.22±0.31 | ??6.20±0.07 | ??2.43±0.03 * | ??3.64±0.03 * | ??307.2±9.1 |
| The true long unit (P﹠B company) that generates | ??29.63±0.15 | ??6.24±0.05 | ??2.31±0.03 | ??3.50±0.05 | ??311.2±9.6 |
| Leaf acyl EFS | ??30.14±0.16 * | ??6.22±0.12 | ??2.31±0.03 | ??3.55±0.05 | ??314.3±7.6 |
*: P＜0.05, Duncan multiple check (Ducan ' s multiple test)
＜example 6〉improve the effect of wrinkle and the effect of healing wounds
Example 6-1: about cell activation and the biosynthetic test of promotion collagen
The present inventor finds that leaf acyl EFS has higher cell activation effect.The implication of term " cell activation effect " comprises the activation of cell growth, activation that meta-bolites produces, for mitochondrial activation in the toxicide cell etc.The cell activation effect can be induced the effect of skin wound healing and be improved the effect of wrinkle.
Identify by this experiment, leaf acyl EFS promotes the biosynthesizing of cell growth and collagen.Used cell is fibroblastic primary culture (people's newborn skin inoblast in this experiment; (humandermal fibroblast neonatal, HDF-N; Gibco, Cooper, people such as M.L., Biomaterials12:243,1991)), and used substratum is newborn infant's fibroblastic growth substratum (FGM, a KDR company).Since leaf acyl EFS comprise incorporate 10% folic acid of having an appointment into polyglutamic acid as main ingredient, so leaf acyl EFS is compared with common folic acid.People's newborn skin inoblast of 1 * 105 cells/well and folic acid or leaf acyl EFS were cultivated 24 hours, taken the photo (referring to Fig. 6) of cultivating product, and test quantitative viable cell (Sigma by toluylene red (NR); People such as Shao-Zeng Z., Cell Biology andToxicology, 6:2,1990) (referring to Fig. 7).
When using leaf acyl EFS, cell growth rate is higher, specifically, when using same concentrations folic acid, can obtain cell growth rate high when only using folic acid.In addition, when only using folic acid and increasing the concentration of folic acid gradually, the not corresponding increase of cell growth rate.Yet, when using leaf acyl EFS and increasing the concentration of leaf acyl EFS gradually, the corresponding obvious change of cell growth rate.Described the possibility of result is derived from folic acid and the water miscible difference of leaf acyl EFS.Specifically, leaf acyl EFS has highly water-soluble, and influences more cell thus.Described promotion cell growth effect causes the effect that improves wrinkle when being used for makeup, and when being used for medicine, causes the effect that promotes wound healing.
Leaf acyl EFS and people's newborn skin inoblast were cultivated 3 days, and separation of supernatant, and end user's 1 Collagen Type VI ELISA test kit (human collagen type-1ELISA kit; Cosmo Bio company) quantitative collagen.Specimen in use is 1% solution that contains composition therefor 2 in the preparation example 2 in this experiment, and with 4%1, the 3-butyleneglycol adds in the sample, and preserves.In addition, for the effect of leaf acyl EFS relatively, identical folic acid solution among preparation folic acid concentration and the leaf acyl EFS.In addition, with 10 nanograms/milliliter transforming growth factor-beta (TGF-β; Sigma company) (being known as the biosynthetic promotion factor of collagen) compares with leaf acyl EFS.
The biosynthetic effect of promotion collagen of leaf acyl EFS is shown in the column diagram of Fig. 8.It as the leaf acyl EFS of cosmetic material manufacturing 1% liquid product.In Fig. 8, and the ratio of the volume of the concentration of x axle (%) expression leaf acyl EFS and the volume of whole culture solution (v/v, %).The result is expressed as when the mean value of collagen is made as 100% in will be not with the group of any mass treatment, and the value of collagen is with respect to the ratio of collagen mean value in the group of not using any mass treatment in each group.Thus, for TGF-β, 0.63% (v/v, %) under, maximum promotion rate is 22%; And for leaf acyl EFS, 0.63% (v/v, %) under, maximum promotion rate is 109%.That is to say that the promotion rate of leaf acyl EFS is 4 times of TGF-β that are applied to comparative group, this shows can be by producing excessive collagen, the effect of the wrinkle that is improved in human skin.Simultaneously, when only using folic acid, the biosynthetic effect of collagen does not appear promoting in limit of error.This shows that leaf acyl EFS can be used as cosmetics material very effectively, particularly, improves the material of wrinkled appearance by the collagen biosynthesizing.
Example 6-2: the test that improves the wrinkle effect of relevant make-up composition
Improve the wrinkle effect for what differentiate leaf acyl EFS, the composition 2 (experimental group) of preparation in the composition 1 (control group) of preparation in the example 2 and the preparation example 2 is applied on 29 30 years old women's the wrinkle of around eyes every day twice, lasting 1 month.After 1 month,, assess the degree that wrinkled appearance improves by with reference to individual questionnaire of being answered and about the analytical results of wrinkle nutrient substance.In the questionnaire that individuality is answered, each problem provides 4 options, before promptly relatively using and when using the back: " do not have and improve ", " less improvement ", " obviously improving " and " improvement clearly ".
By image analysis assessment wrinkle.Before carrying out test, use Visioscan SV600 (CK company) to collect the replica (replica) of eyes below, and when test is finished, collect replica, measure wrinkle density by image analysis from same position.The result is expressed as the wrinkle decrement.
The results are shown in the table 5, and as can be seen, when the composition 2 that will comprise leaf acyl EFS compared with composition 1, wrinkle alleviated to some extent, and its outward appearance makes moderate progress.In addition, when using the composition 2 that comprises leaf acyl EFS, with use before compare, wrinkle density is reduced to about 45%.
Described result shows, when composition of the present invention is used as the external preparation for skin product, can effectively improve the outward appearance of the wrinkle that forms in the skin.This shows, can be with leaf acyl EFS effectively as cosmetics material.
The improvement degree and the density of the wrinkle of skin that table 5. make-up composition causes
| Do not have and improve (number) | The less improvement (number) | Obviously improve (number) | Improving clearly (number) | Wrinkle density (%) | |
| |
?25 | ?3 | ?1 | ??0 | ??95 |
| Composition 2 | ?0 | ?4 | ?10 | ??15 | ??45 |
＜example 7〉suppress effect by ultraviolet ray (UV) radiation-induced cell injury
In this experiment, the present inventor identifies, and leaf acyl EFS suppresses by the radiation-induced cell injury of UV.
In human keratinocyte's growth medium (KGM, KDR company), cultivate 1 * 10
5Human keratinocyte's primary culture of individual cells/well is the leaf acyl EFS of about 10 weight % together with the folic acid amount, continues about 72 hours, is the cell predetermined amount of time of the UV radiation exposure growth of 306 nanometers subsequently with wavelength.Can control the concentration of folic acid in the end product by adjusting the concentration of in leaf acyl EFS, incorporating folic acid between yeast phase into.Behind irradiation UV ray, cultivated the gained culture about 24 hours, and come quantitative viable cell by the NR test.Irradiation time is defined as the time of about 50% necrocytosis in preliminary experiment.The result is explained as follows:
Survival rate (%)=(growth velocity of each group behind the UV radiation exposure)/(not being exposed to the growth velocity that the UV radiating has the group of same concentrations)
In the substratum of the various concentration leaf acyl EFS of growth, proportional to the concentration of the inhibition of the radiation-induced cell injury of UV and institute's grown cell.Different with the result who promotes the cell growth test in the example 6, when being 0.62%, folic acid concentration obtains high viability.Although this possibility of result is because the growth velocity of the cell of growing in the substratum that comprises about 0.62% leaf acyl EFS is lower than the growth velocity of the cell of growing in the substratum that is comprising about 0.31% leaf acyl EFS, but when using about 0.62% leaf acyl EFS, the inhibiting rate of UV inductive cell injury is higher than inhibiting rate when using about 0.31% leaf acyl EFS.This shows, when adding to leaf acyl EFS in the makeup, effectively shielding of ultraviolet (UV) radiation.Specifically, use about 0.1% to 6% leaf acyl EFS to increase the UV barrier effect of sunscreen product, also help to increase the UV barrier effect of conventional formulation (for example emulsion and breast frost) except that helping.
Claims (25)
1. leaf acyl fermented soybean extract, it comprises the folic acid polyglutamic acid as main ingredient, wherein said leaf acyl fermented soybean extract is to produce by the culture fermentation of using microorganism to make to comprise folic acid and soybean extraction.
2. leaf acyl fermented soybean extract according to claim 1 is characterized in that described microorganism is selected from bacillus micro-organism or Corynebacterium microorganism.
3. leaf acyl fermented soybean extract according to claim 1 is characterized in that described microorganism is a subtilis.
4. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has antihistamine effect.
5. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has anti-allergic effects.
6. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has the effect that promotes calcium absorption.
7. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has the effect that promotes bone growth.
8. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has the biosynthetic effect of the collagen of promotion.
9. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has the effect that improves wrinkle.
10. according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3, it is characterized in that described leaf acyl fermented soybean extract has the effect that promotes the cell growth.
11. a composition that is used for external preparation for skin product or makeup, it comprises according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3 as active ingredient.
12. the composition that is used for external preparation for skin product or makeup according to claim 11 is characterized in that, described composition is used for antianaphylaxis or improves atopic dermatitis.
13. the composition that is used for external preparation for skin product or makeup according to claim 11 is characterized in that, described composition be used to activate skin cells metabolism, prevent that wrinkle from forming or improving wrinkled appearance.
14. the composition that is used for external preparation for skin product or makeup according to claim 11 is characterized in that, described composition is used to suppress the cell injury that caused by ultraviolet radiation.
15. a health care supplementing food composition, it comprises according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3 as active ingredient.
16. health care supplementing food composition according to claim 15 is characterized in that, described health care supplementing food composition is to be used for drink.
17. health care supplementing food composition according to claim 15 is characterized in that, described health care supplementing food composition has the effect that promotes growth.
18. health care supplementing food composition according to claim 15 is characterized in that, the effect that described health care supplementing food composition has the osteoporosis of preventing.
19. health care supplementing food composition according to claim 15 is characterized in that, described health care supplementing food composition has the effect that promotes calcium absorption.
20. health care supplementing food composition according to claim 15 is characterized in that, described health care supplementing food composition has the allergy of preventing or improves the effect of physique.
21. a feed composition, it comprises according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3 as active ingredient.
22. a promotion comprises the medicinal compositions of people in the growth of interior animal, it comprises according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3 as active ingredient.
23. the treatment or the medicinal compositions of preventing osteoporosis disease, it comprises according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3 as active ingredient.
24. a medicinal compositions that promotes calcium absorption, it comprises according to the described leaf acyl of arbitrary claim fermented soybean extract in the claim 1 to 3 as active ingredient.
25. a method of making leaf acyl fermented soybean extract, it is by the use microorganism culture fermentation that comprises folic acid and soybean extraction to be carried out.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020070100311A KR100926170B1 (en) | 2007-10-05 | 2007-10-05 | Biopolymer produced by fermenting the extract of soybean with folic acid and a composition containing thereof |
| KR10-2007-0100311 | 2007-10-05 | ||
| PCT/KR2008/005816 WO2009045064A1 (en) | 2007-10-05 | 2008-10-02 | Biopolymer produced by fermenting the extract of soybean with folic acid and a composition containing thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101883860A true CN101883860A (en) | 2010-11-10 |
| CN101883860B CN101883860B (en) | 2013-12-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008801188773A Active CN101883860B (en) | 2007-10-05 | 2008-10-02 | Biopolymer produced by fermenting extract of soybean with folic acid and composition containing thereof |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US8431364B2 (en) |
| JP (1) | JP5270680B2 (en) |
| KR (1) | KR100926170B1 (en) |
| CN (1) | CN101883860B (en) |
| WO (1) | WO2009045064A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105105031A (en) * | 2015-09-23 | 2015-12-02 | 昆明理工大学 | Method for producing fermented soybean rich in folic acid |
| CN109431880A (en) * | 2018-12-05 | 2019-03-08 | 广州润虹医药科技股份有限公司 | A kind of whitening maintenance agent and the preparation method with whitening repair function sleep mask |
| CN109984977A (en) * | 2019-05-21 | 2019-07-09 | 上海乾一化学品有限公司 | A kind of Essence of the glycogen containing plant and preparation method thereof |
| CN110882195A (en) * | 2019-12-23 | 2020-03-17 | 广州倍健医疗用品有限公司 | Collagen moisturizing and anti-wrinkle composition and preparation method thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120190745A1 (en) * | 2009-09-30 | 2012-07-26 | Shiseido Company. Ltd. | Oral composition for reducing wrinkle formation |
| BR112013001369B1 (en) * | 2010-07-20 | 2022-03-22 | Microbio Co., Ltd | Use of a fermented soy extract and composition |
| KR101533067B1 (en) * | 2013-05-14 | 2015-07-01 | 가천대학교 산학협력단 | Composition for improving skin wrinkle and skin moisturing comprising Soybean fermented by Pichia jadinii and Aureobasidium pullulans bacteria |
| US20140363504A1 (en) * | 2013-06-11 | 2014-12-11 | The Beauty Nation Ptd Ltd | Absorbable encapsulated calcium in hypromellose capsule |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002026961A2 (en) * | 2000-09-29 | 2002-04-04 | Showa Denko K.K. | Production process of surfactin |
| DE60134386D1 (en) * | 2001-03-21 | 2008-07-24 | Microbio Co Ltd | A method for inhibiting cancer growth with a fermented soy extract |
| US20020182274A1 (en) | 2001-03-21 | 2002-12-05 | Kung-Ming Lu | Methods for inhibiting cancer growth, reducing infection and promoting general health with a fermented soy extract |
| WO2002080862A1 (en) | 2001-04-06 | 2002-10-17 | Toyo Hakko Co., Ltd. | Cosmetic materials and process for producing the same |
| CH696628A5 (en) * | 2002-02-26 | 2007-08-31 | Eprova Ag | Use of folate suitable for the manufacture of a preparation for the prevention and treatment of inflammation and inflammation-associated diseases, in particular for influencing the |
| JP4484014B2 (en) * | 2002-03-07 | 2010-06-16 | ヒガシマル醤油株式会社 | Hyaluronidase inhibition, antiallergic activity and immunostimulatory substances |
| KR20050094081A (en) * | 2004-03-22 | 2005-09-27 | 주식회사 안국약품식품사업부 | Pharmaceutical composition comprising the viscos substances extracted from chongkukjang for fibrinolysis |
| JP2006001922A (en) * | 2004-05-20 | 2006-01-05 | Mitsukan Group Honsha:Kk | Allergy inhibitory composition |
| JP4137062B2 (en) * | 2005-01-12 | 2008-08-20 | トン ハイ バイオテクノロジー コーポレイション | Γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate and γ-polyglutamate hydrogels for use as super humectants in cosmetics and personal care products |
| JP5128760B2 (en) * | 2005-05-16 | 2013-01-23 | トン ハイ バイオテクノロジー コーポレイション | Γ-polyglutamic acid (γ-PGA, H form) and γ-polyglutamate for use as dietary supplements in dietary products |
| KR20060124466A (en) * | 2005-05-31 | 2006-12-05 | 주식회사 창성테크 | Vitamin beverage including of fermented soybeans and preparation method of thereof |
| WO2007002572A2 (en) * | 2005-06-24 | 2007-01-04 | N-Zymeceuticals, Inc. | Nattokinase for reducing whole blood viscosity |
| KR20080047358A (en) * | 2005-09-22 | 2008-05-28 | 가부시키가이샤 시세이도 | Wrinkle-improving agent |
-
2007
- 2007-10-05 KR KR1020070100311A patent/KR100926170B1/en active IP Right Grant
-
2008
- 2008-10-02 WO PCT/KR2008/005816 patent/WO2009045064A1/en active Application Filing
- 2008-10-02 JP JP2010527888A patent/JP5270680B2/en active Active
- 2008-10-02 CN CN2008801188773A patent/CN101883860B/en active Active
- 2008-10-02 US US12/681,725 patent/US8431364B2/en active Active
-
2013
- 2013-03-15 US US13/834,402 patent/US9084748B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105105031A (en) * | 2015-09-23 | 2015-12-02 | 昆明理工大学 | Method for producing fermented soybean rich in folic acid |
| CN109431880A (en) * | 2018-12-05 | 2019-03-08 | 广州润虹医药科技股份有限公司 | A kind of whitening maintenance agent and the preparation method with whitening repair function sleep mask |
| CN109984977A (en) * | 2019-05-21 | 2019-07-09 | 上海乾一化学品有限公司 | A kind of Essence of the glycogen containing plant and preparation method thereof |
| CN110882195A (en) * | 2019-12-23 | 2020-03-17 | 广州倍健医疗用品有限公司 | Collagen moisturizing and anti-wrinkle composition and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20090035180A (en) | 2009-04-09 |
| CN101883860B (en) | 2013-12-25 |
| US8431364B2 (en) | 2013-04-30 |
| US20110288025A1 (en) | 2011-11-24 |
| WO2009045064A1 (en) | 2009-04-09 |
| JP5270680B2 (en) | 2013-08-21 |
| JP2010540623A (en) | 2010-12-24 |
| US9084748B2 (en) | 2015-07-21 |
| KR100926170B1 (en) | 2009-11-10 |
| US20130210737A1 (en) | 2013-08-15 |
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Effective date of registration: 20161208 Address after: Seoul, South Korea Patentee after: Le Baiyou Corporation Patentee after: Jin Guangnian Address before: Seoul, South Korea Patentee before: Damy Chemical Co. Ltd Patentee before: Jin Guangnian |